This proposal is focused on determining the mechanism of peroxisomal protein import mediated by the receptor, PEX5. The inability to import proteins into the peroxisomal matrix is responsible for many serious diseases. Mutations in PEX5 have been shown to cause Zellweger Syndrome, a cerebro-hepato-renal dysfunction that causes death. We propose to investigate the underlying structural, kinetic, and thermodynamic determinants that give rise to such phenotypes. Such studies are crucial in understanding the molecular basis of all PEX5 related disorders. PEX5 is a 68 kDa protein that recognizes the tripeptide peroxisomal targeting signal-1 (PTS1). We will (1) study the kinetics and thermodynamics of PEX5-PTS 1 complex formation using fluorescence anisotropy; (2) use site-directed mutagenesis to determine which amino acids in PEX5 are involved in signal recognition; (3) use CD spectroscopy, X-ray crystallography and analytical ultracentrifugation to study the secondary, tertiary, and quaternary structural changes in PEX5 that alter its specificity for other membrane-associated peroxins; and (4) test our hypotheses about PTS 1 recognition and import using PEX5- deficient cells in an in vivo assay.